1. Field of the Invention
The present invention relates to methods for separating two or more kinds of molecules using dielectrophoretic forces.
2. Background of the Invention
Recently, advance in semiconductor technology has established processing technology of materials at scales of nanometer to micrometer by means of micromachining technology such as photolithography, which continues to make advance also at the present.
In the fields of chemistry and biochemistry, new technology called a Micro Total Analysis System (μ-TAS), Laboratory on a chip is growing, in which such micromachining technology is employed to carry out a whole series of chemical/biochemical analytical steps of extraction of component(s) to be analyzed from biological samples (extraction step), analysis of the component(s) with chemical/biochemical reaction(s) (analysis step), and subsequent separation (separation step) and detection (detection step) using a highly small analytical device integrated on a chip having each side of a few centimeters to a few ten centimeters in length.
Procedures of the μ-TAS are expected to make a large contribution to saving the analyzing time, reducing the amounts of samples to be used and reagents for chemical/biochemical reactions, and reducing the size of analytical instruments and the space for analysis in the course of all the chemical/biochemical analytical steps.
For the separation step in μ-TAS, in particular, there have been developed capillary electrophoretic methods in which a capillary (fine tube) with an inner diameter of less than 1 mm which is made of Teflon, silica, or the like as material is used as the separating column to achieve separation with charge differences of substances under a high electric field, and capillary column chromatographic methods in which a similar capillary is used to achieve separation with the difference of the interaction between carrier in the column medium and substances.
However, capillary electrophoretic methods need a high voltage for separation and have a problem of a low sensitivity of detection due to a limited capillary volume in the detection area and also these is found such a problem that they are not suitable for separation of high molecular weight substances, though suitable for separation of low molecular weight substances, since the length of capillary for separation is limited on the capillary chip on a chip and thus a capillary can not be made into a length enough for separating high molecular weight substances. In addition, in capillary column chromatographic methods there is a limit in making the throughput of separation processing higher and also these is such a problem that reducing the processing time is difficult.
Thus, one means to solve problems as described above are now noticed separation methods utilizing the phenomenon in which the placement of substances under a nonuniform electric field results in the positive and negative polarization within the substances, thereby providing a driving force of moving the substances, so-called dielectrophoretic force [H. A. Pohl, “Dielectrophoresis”, Cambridge Univ. Press (1978); T. B. Jones, “Electromechanics of Particles”, Cambridge Univ. Press (1995), and the like].
These separation methods are presently believed to be the most suitable separation method in m-TAS from the following points: (1) a rapid separation can be expected at a low applied voltage without requiring a high voltage as in capillary electrophoresis, since an electric field and its gradient can be increased to an extreme extent if micromachined electrodes are employed, because the degree of dielectrophoretic forces depends on the size and dielectric properties of substances (particles) and is proportional to the electric field gradient; (2) an increase in temperature due to applying the electric field can be minimized, and a high electric field can be formed, since a strong electric field area is localized at a significantly small region; (3) as the dielectrophoretic force is a force proportional to the electric field gradient, the force is understood as independent on the polarity of the applied voltage, and thus works under an AC electric field in a similar way to a D.C. electric field, and therefore if a high frequency A.C is employed, an electrode reaction (electrolytic reaction) in an aqueous solution can be suppressed, so that the electrodes themselves can be integrated in the channel (sample flow path); (4) improvement in a detection sensitivity can be expected, since there is no restriction to a chamber volume of the detection component as in capillary electrophoresis, and the like.
As separation methods utilizing dielectrophoretic forces as described above, there have been reported various methods until now [M. Washizu, et. al., IEEE Transaction IA, vol. 30, No. 4, pp. 835–843 (1994); M. Washizu, et. al., Conf. Rec. The Institute of Electrostatics Japan, '93 Ann. Meet. (Int'l Session), pp. 27–32 (1993); Y. Huang, et al., Biophys. J., vol. 73, pp. 1118–1129 (1997); and N. G. Green et al., J. Phys. D.: Appl. Phys. vol. 31, 25–30 (1998), and the like].
For example, Journal of Physics D, British Journal of Applied Physics (J. Phys. D: Appl. Phys.), 27, 2659–2662 (1994) describes that from suspensions containing HL-60 cells and normal blood cells, respective cells can be separated; Microbiology, 140, 585–591 (1994) describes that from suspensions containing different microorganisms, the microorganisms can be separated into different species of yeast and bacteria from one another; Journal of Biotechnology, 32, 29–37 (1994) describes that from suspensions containing living and dead cells of yeast, both cells can be separated from each other; and J. Phys. D: Appl. Phys., 31, 25–30 (1998) describes that from suspensions containing latex particles having a diameter of 93 nm and 216 nm, both particles can be separated by dielectrophoresis and electro-fluid forces from each other.
Additionally, M. Washizu, et al., IEEE Transaction IA, vol. 30, No. 4, pp. 835–843 (1994) reported that using solutions containing a single biological component as samples, the component for example, avidin (68 kDa), concanavalin A (52 kDa), chymotrypsinogen A (25 kDa), or ribonuclease A (13.7 kDa)] is captured on the electrode by dielectrophoretic forces, and also using solutions containing a single biological component as samples, the component can be captured on the electrode by dielectrophoretic forces [the capture ratio was 100% when using a sample of 48.5 kb DNA alone, about 60% when using a sample of 15 kb DNA alone, about 50% when using a sample of 9 kb circular DNA alone, and a few % using a sample of avidin (68 kDa) alone].
However, reports on separation methods with conventional dielectrophoretic forces as described above are limited to separating particles having a low solubility in a solution, relative to DNAs and proteins, such as various cells and latex particles, or otherwise only capturing a single (one kind of) DNA or protein, and any report has not been presented yet on separation of respective molecules from solutions in which are dissolved two or more kinds of biological component molecules, in particular, such as for example DNAs and proteins.
This is because two kinds or more of molecules such as proteins and DNAs, which have a very small physical size, as compared with cells and latex particles, are considered to be difficult in separation from each other from solution in which those molecules are dissolved on the basis of the difference between the size of respective molecules by using dielectrophoretic forces, since the strength of dielectrophoretic forces depends on the physical size of substances, so that substances having a larger volume will receive a larger dielectrophoretic force, and also because conventional separation has been carried out at a weak electric field strength lower than 500 KV/m, whereby separation is not achievable.